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Publication Open Access A cartridge based sensor array platform for multiple coagulation measurements from plasma(Royal Society of Chemistry (RSC), 2015) Bulut, Serpil; Yaralioglu, G. G.; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Çakmak, Onur; Ermek, Erhan; Kılınç, Necmettin; Barış, İbrahim; Kavaklı, İbrahim Halil; Ürey, Hakan; PhD Student; Other; Researcher; Teaching Faculty; Faculty Member; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; College of Engineering; Graduate School of Sciences and Engineering; College of Sciences; N/A; 109991; N/A; 111629; 40319; 8579This paper proposes a MEMS-based sensor array enabling multiple clot-time tests for plasma in one disposable microfluidic cartridge. The versatile LoC (Lab-on-Chip) platform technology is demonstrated here for real-time coagulation tests (activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT)). The system has a reader unit and a disposable cartridge. The reader has no electrical connections to the cartridge. This enables simple and low-cost cartridge designs and avoids reliability problems associated with electrical connections. The cartridge consists of microfluidic channels and MEMS microcantilevers placed in each channel. The microcantilevers are made of electroplated nickel. They are actuated remotely using an external electro-coil and the read-out is also conducted remotely using a laser. The phase difference between the cantilever oscillation and the coil drive is monitored in real time. During coagulation, the viscosity of the blood plasma increases resulting in a change in the phase read-out. The proposed assay was tested on human and control plasma samples for PT and aPTT measurements. PT and aPTT measurements from control plasma samples are comparable with the manufacturer's datasheet and the commercial reference device. The measurement system has an overall 7.28% and 6.33% CV for PT and aPTT, respectively. For further implementation, the microfluidic channels of the cartridge were functionalized for PT and aPTT tests by drying specific reagents in each channel. Since simultaneous PT and aPTT measurements are needed in order to properly evaluate the coagulation system, one of the most prominent features of the proposed assay is enabling parallel measurement of different coagulation parameters. Additionally, the design of the cartridge and the read-out system as well as the obtained reproducible results with 10 mu l of the plasma samples suggest an opportunity for a possible point-of-care application.
Publication Metadata only CRISPR-CAS13 system as a promising and versatile tool for cancer diagnosis, therapy, and research(American Chemical Society (ACS), 2021) Palaz, Fahreddin; Kalkan, Ali Kerem; Demir, Ayca Nur; Tozluyurt, Abdullah; Ozcan, Ahsen; Ozsoz, Mehmet; Department of Molecular Biology and Genetics; Can, Özgür; Undergraduate Student; Department of Molecular Biology and Genetics; College of Sciences; N/AOver the past decades, significant progress has been made in targeted cancer therapy. In precision oncology, molecular profiling of cancer patients enables the use of targeted cancer therapeutics. However, current diagnostic methods for molecular analysis of cancer are costly and require sophisticated equipment. Moreover, targeted cancer therapeutics such as monoclonal antibodies and small-molecule drugs may cause off-target effects and they are available for only a minority of cancer driver proteins. Therefore, there is still a need for versatile, efficient, and precise tools for cancer diagnostics and targeted cancer treatment. In recent years, the CRISPR-based genome and transcriptome engineering toolbox has expanded rapidly. Particularly, the RNA-targeting CRISPR-Cas13 system has unique biochemical properties, making Cas13 a promising tool for cancer diagnosis, therapy, and research. Cas13-based diagnostic methods allow early detection and monitoring of cancer markers from liquid biopsy samples without the need for complex instrumentation. In addition, Cas13 can be used for targeted cancer therapy through degrading and manipulating cancer-associated transcripts with high efficiency and specificity. Moreover, Cas13-mediated programmable RNA manipulation tools offer invaluable opportunities for cancer research, identification of drug-resistance mechanisms, and discovery of novel therapeutic targets. Here, we review and discuss the current use and potential applications of the CRISPR-Cas13 system in cancer diagnosis, therapy, and research. Thus, researchers will gain a deep understanding of CRISPR-Cas13 technologies, which have the potential to be used as next-generation cancer diagnostics and therapeutics.Publication Metadata only Development and validation of a cost-effective in-house method, tetra-primer ARMS PCR assay, in genotyping of seven clinically important point mutations(Academic Press Ltd- Elsevier Science Ltd, 2011) Etlik, Ozdal; Koksal, Vedat; Arican-Baris, S. Tugba; Department of Molecular Biology and Genetics; Barış, İbrahim; Teaching Faculty; Department of Molecular Biology and Genetics; College of Sciences; 111629The single nucleotide polymorphism (SNP) genotyping is currently considered as a particularly valuable tool for the diagnosis of different pathologies. For this reason, over the past several years a great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. Although a large number of distinct approaches has been reported each laboratory use one of the published methods based on their technical and economical capacity. This article presents an application of an in-house assay, tetra-primer ARMS PCR assay, and its application in SNP genotyping. We have shown that this assay could be more advantageous when compared with PCR-RFLP, real time PCR, and DNA sequencing. We have shown that the assay is successful in genotyping using archived paraffin-embedded tissues, heparinated samples and amniotic fluids with meconium. These low-costed (3$/reaction) assays could be completed within 3-4 h after specimen receipt allowing for a reasonable turn-around time in the laboratory. Since tetra-primer ARMS PCR assay does not require any special equipment, the assay could be set up in most clinical diagnostic laboratories. (C) 2011 Elsevier Ltd. All rights reserved.Publication Metadata only Isolation and characterization of cDNAs of lentil ADP-glucose pyrophosphorylase(Current Biology Ltd, 2011) Oz, Gul Cevahir; Tulum, Isil; Department of Chemical and Biological Engineering; Department of Molecular Biology and Genetics; Kavaklı, İbrahim Halil; Barış, İbrahim; Faculty Member; Teaching Faculty; Department of Chemical and Biological Engineering; Department of Molecular Biology and Genetics; College of Engineering; College of Sciences; 40319; 111629N/APublication Metadata only Labeling carboxyl groups of surface-exposed proteins provides an orthogonal approach for cell surface isolation(Amer Chemical Soc, 2018) Tan, Edwin; Mitchison, Timothy; N/A; N/A; Department of Molecular Biology and Genetics; Küçük, Nazlı Ezgi Özkan; Şanal, Erdem; Researcher; PhD Student; Faculty Member; Department of Molecular Biology and Genetics; N/A; Graduate School of Sciences and Engineering; College of Sciences; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); N/A; N/A; 105301Quantitative profiling of cell surface proteins is critically important for the understanding of cell-cell communication, signaling, tissue development, and homeostasis. Traditional proteomics methods are challenging for cell surface proteins due to their hydrophobic nature and low abundance, necessitating alternative methods to efficiently identify and quantify this protein group. Here we established carboxyl reactive biotinylation for selective and efficient biotinylation and isolation of surface-exposed proteins of living cells. We assessed the efficiency of carboxyl-reactive biotinylation for plasma membrane proteins by comparing it with a well-established protocol, amine-reactive biotinylation, using SILAC (stable isotope labeling in cell culture). Our results show that carboxyl-reactive biotinylation of cell surface proteins is both more selective and more efficient than amine-reactive biotinylation. We conclude that it is a useful approach, which is partially orthogonal to amine-reactive biotinylation, allowing us to cast a wider net for a comprehensive profiling of cell surface proteins.Publication Metadata only MEMS biosensor for blood plasma viscosity measurements(Elsevier Science Bv, 2012) N/A; Department of Electrical and Electronics Engineering; Department of Mechanical Engineering; N/A; N/A; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Department of Mechanical Engineering; Department of Electrical and Electronics Engineering; Çakmak, Onur; Elbüken, Çağlar; Ermek, Erhan; Bulut, Selma; Kılınç, Yasin; Barış, İbrahim; Kavaklı, İbrahim Halil; Alaca, Burhanettin Erdem; Ürey, Hakan; PhD Student; Researcher; Other; PhD Student; PhD Student; Teaching Faculty; Faculty Member; Faculty Member; Faculty Member; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Department of Mechanical Engineering; Department of Electrical and Electronics Engineering; Koç University Surface Science and Technology Center (KUYTAM) / Koç Üniversitesi Yüzey Teknolojileri Araştırmaları Merkezi (KUYTAM); Graduate School of Sciences and Engineering; College of Engineering; College of Engineering; Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; College of Sciences; College of Engineering; College of Engineering; College of Engineering; N/A; N/A; N/A; N/A; N/A; 111629; 40319; 115108; 8579N/APublication Metadata only Microcantilever based disposable viscosity sensor for serum and blood plasma measurements(Academic Press Inc Elsevier Science, 2013) N/A; Department of Mechanical Engineering; Department of Mechanical Engineering; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Department of Mechanical Engineering; Department of Chemical and Biological Engineering; Çakmak, Onur; Elbüken, Çağlar; Ermek, Erhan; Mostafazadeh, Aref; Barış, İbrahim; Alaca, Burhanettin Erdem; Kavaklı, İbrahim Halil; Ürey, Hakan; PhD Student; Researcher; Faculty Member; Researcher; Teaching Faculty; Faculty Member; Faculty Member; Faculty Member; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Department of Mechanical Engineering; Department of Chemical and Biological Engineering; Graduate School of Sciences and Engineering; College of Engineering; College of Engineering; Graduate School of Sciences and Engineering; College of Sciences; College of Engineering; College of Engineering; N/A; N/A; N/A; N/A; 111629; 115108; 40319; 8579This paper proposes a novel method for measuring blood plasma and serum viscosity with a microcantilever-based MEMS sensor. MEMS cantilevers are made of electroplated nickel and actuated remotely with magnetic field using an electro-coil. Real-time monitoring of cantilever resonant frequency is performed remotely using diffraction gratings fabricated at the tip of the dynamic cantilevers. Only few nanometer cantilever deflection is sufficient due to interferometric sensitivity of the readout. The resonant frequency of the cantilever is tracked with a phase lock loop (PLL) control circuit. The viscosities of liquid samples are obtained through the measurement of the cantilever's frequency change with respect to a reference measurement taken within a liquid of known viscosity. We performed measurements with glycerol solutions at different temperatures and validated the repeatability of the system by comparing with a reference commercial viscometer. Experimental results are compared with the theoretical predictions based on Sader's theory and agreed reasonably well. Afterwards viscosities of different Fetal Bovine Serum and Bovine Serum Albumin mixtures are measured both at 23 degrees C and 37 degrees C, body temperature. Finally the viscosities of human blood plasma samples taken from healthy donors are measured. The proposed method is capable of measuring viscosities from 0.86 cP to 3.02 cP, which covers human blood plasma viscosity range, with a resolution better than 0.04 cP. The sample volume requirement is less than 150 mu l and can be reduced significantly with optimized cartridge design. Both the actuation and sensing are carried out remotely, which allows for disposable sensor cartridges. (C) 2013 Published by Elsevier Inc.Publication Metadata only Phosphoproteomic analysis of aurora kinase inhibition in monopolar cytokinesis(Amer Chemical Soc, 2015) Giese, Sven H.; Hu, Chi-Kuo; Renard, Bernhard Y.; N/A; N/A; N/A; N/A; Department of Molecular Biology and Genetics; Köken, Ayşe Nur Polat; Karayel, Özge; Harmanda, Büşra; Şanal, Erdem; Master Student; Master Student; Researcher; Master Student; Faculty Member; Department of Molecular Biology and Genetics; Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; College of Sciences; 239008; N/A; N/A; N/A; 105301Cytokinesis is the last step of the cell cycle that requires coordinated activities of the microtubule c-ytoskeleton, actin cytoskeleton, and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (stable isotope labeling with amino acids in cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680- or AZD1152-mediated aurora kinase inhibition. In total, our analysis quantified over 20 000 phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.Publication Metadata only Proteomics in cell division(Wiley, 2017) N/A; N/A; N/A; N/A; N/A; Department of Molecular Biology and Genetics; Kagiali, Zeynep Cansu Üretmen; Şentürk, Aydanur; Küçük, Nazlı Ezgi Özkan; Qureshi, Mohammad Haroon; PhD Student; PhD Student; Researcher; PhD Student; Faculty Member; Department of Molecular Biology and Genetics; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; N/A; Graduate School of Sciences and Engineering; College of Sciences; N/A; N/A; N/A; N/A; 105301Cell division requires a coordinated action of the cell cycle machinery, cytoskeletal elements, chromosomes, and membranes. Cell division studies have greatly benefitted from the mass spectrometry (MS)-based proteomic approaches for probing the biochemistry of highly dynamic complexes and their coordination with each other as a cell progresses into division. In this review, the authors first summarize a wide-range of proteomic studies that focus on the identification of sub-cellular components/protein complexes of the cell division machinery including kinetochores, mitotic spindle, midzone, and centrosomes. The authors also highlight MS-based large-scale analyses of the cellular components that are largely understudied during cell division such as the cell surface and lipids. Then, the authors focus on posttranslational modification analyses, especially phosphorylation and the resulting crosstalk with other modifications as a cell undergoes cell division. Combining proteomic approaches that probe the biochemistry of cell division components with functional genomic assays will lead to breakthroughs toward a systems-level understanding of cell division.Publication Metadata only Proximal biotinylation-based combinatory approach for isolating integral plasma membrane proteins(Amer Chemical Soc, 2020) N/A; Department of Molecular Biology and Genetics; N/A; N/A; N/A; Department of Molecular Biology and Genetics; Akdağ, Mehmet; Yunt, Zeynep Sabahat; Kamacıoğlu, Altuğ; Qureshi, Mohammad Haroon; Akarlar, Büşra; Master Student; Teaching Faculty; Master Student; PhD Student; Other; Faculty Member; Department of Molecular Biology and Genetics; Graduate School of Sciences and Engineering; College of Sciences; Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; N/A; College of Sciences; N/A; 116178; N/A; N/A; N/A; 105301Comprehensive profiling of the cell-surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra in vitro labeling step, which we call Plasma Membrane-BiolD. The lipid-modified BirA* enzyme (MyrPalm BirA*) was targeted to the inner leaflet of the plasma membrane, where it effectively biotinylated plasma membrane proteins. Biotinylated proteins were then affinity-purified and analyzed by mass spectrometry. Our analysis demonstrates that combining conventional sucrose density gradient centrifugation and Plasma Membrane-BioID is ideal to overcome the inherent limitations of the identification of integral membrane proteins, and it yields highly pure plasma components for downstream proteomic analysis.